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The ability of a series of synthetic analogues of omega-conopeptides MVIIA (SNX-111) and TVIA (SNX-185) to prevent electrically-evoked norepinephrine release from rat tail artery and hippocampal slice preparations was determined in an effort to identify voltage-sensitive calcium channel (VSCC) blockers that selectively target N-type VSCCs in central nervous system tissue. Electrical field stimulation (3 Hz, 1 ms in duration. 80 V for 1 min) caused a high and consistent tritium outflow from rat tail artery and hippocampal slice preparations preloaded with [3H]-norepinephrine. All conopeptides, chosen for their selective affinities for high-affinity SNX-111 binding sites (i.e., N-type VSCCs) over high-affinity omega-conopeptides MVIIC (SNX-230) binding sites (i.e., P/Q-type VSCCs), produced a concentration-dependent inhibition of calcium dependent electrically-evoked tritium outflow from both tail arteries and hippocampal slices: IC50s ranged from 1.2 nM to 1.2 microM. Blocking potencies (IC50s) in the tail artery assay were significantly correlated with those measured in the hippocampal slice preparation (r = 0.91, P = 0.00000012). There was a significant correlation between IC50s for blockade of hippocampal norepinephrine release and the inhibition of high-affinity [125I]-SNX-I11 binding in rat brain synaptosomes (r = 0.76, P = 0.00028). Blockade of hippocampal norepinephrine release was not significantly correlated with the inhibition of high-affinity SNX-230 binding (r = 0.46, P = 0.056). Maximum inhibition of tritium outflow in the tail artery assay was 22+/-1.4% of control, approximating the value (20.9+/-16.0% of control) obtained in the absence of extracellular Ca2+. In contrast, the maximum inhibition of tritium release from hippocampal slices was 36.8+/-2.5% of control (P0.05, compared to that of the tail artery assay). These results suggest that (1) N-type VSCCs alone mediate low frequency electrical stimulation-evoked neurotransmitter release from peripheral sympathetic efferents (tail artery) while both N-type and non-N type(s) mediate neurotransmitter release from CNS neurons (hippocampus); and (2) analogues of omega-conopeptides MVIIA and TVIA do not differentiate between N-type VSCCs mediating norepinephrine release from central and peripheral neural tissues.

Forty years have transpired since tetraethylammonium was first used to selectively inhibit the potassiuin conductance in squid axons. Since then, a large body of work has emerged describing inhibitors of voltage-gated potassium currents in a variety of cells. The advent of molecular cloning techniques and the cloning of the potassium channel encoded by the Shaker locus in Drosophila has enabled detailed structure­ function studies of several potassium channel subunits. These breakthroughs have also recently enabled studies of the "toxinology" and pharmacology of specific potassium channel subunits expressed heterologously in Xenopus oocytes and other cells. Here we describe the results of some of those efforts, focusing in particular on our work with four members of the Shaker subfamily of potassium channel a-subunits: Kvl.1 through Kvl.4. These subunits are expressed in the central nervous system and other tissues of rodents, and are highly homologous to corresponding subunits expressed in humans. We provide a profile of potency and selectivity for.five snake dendrotoxins as well as several scorpion toxins for these potassium channel subunits expressed in Xenopus oocytes. We also provide similar data for four other peptide toxins and several nonpeptide compounds that had previously been shown to inhibit potassium currents. We discuss several potential clinical applications of potassium channel inhibitors, including demyelinating diseases such as multiple sclerosis, immunosuppression, cardiac arrhythmias, neurodegenerative and psychiatric diseases. Further progress will require, among other things, a greater understanding of the expression patterns of potassium channel subunits in the CNS and elsewhere as well as knowledge of the specific subunit composition of heteromultimeric channels.

Several novel N-type voltage sensitive calcium channel blockers showed high affinity in the IMR32 assay and efficacy in the anti-writhing model. Herein, we describe the design, synthesis, SAR studies, biological data, physicochemical properties and pharmacokinetics of this 4-piperidinylaniline series.

Exploration of the SAR around the leucine side chain in a series of N,N-dialkyldipeptidylamines with potent functional activity at N-type VSCC is presented. A novel analog is disclosed which possesses improved aqueous solubility, in vivo activity in an audiogenic seizure model, and reversible blockade in electrophysiological assays.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

This investigation assessed the ability of a variety of calcium channel blocking peptides to block synaptic transmission in the isolated mouse phrenic nerve-hemidiaphragm. The synthetic version of the naturally occurring N-type voltage-sensitive calcium channel (VSCC) blocker omega-conopeptide MVIIA (SNX-111) had no effect on nerve-evoked muscle contractions. The non-N-, non-L-type VSCC blocker, omega-conopeptide MVIIC (SNX-230), blocked neuromuscular transmission completely, as did the selective P-type VSCC blocker, omega-Aga-IVA. Subsequent evaluation of other synthetic omega-conopeptides and analogs disclosed a significant positive correlation between the test compounds' affinities for high-affinity SNX-230 brain binding sites and their neuromuscular blocking potencies. Quantal analysis of transmitter release showed that SNX-230 abolished evoked endplate potentials completely, but had little effect on the amplitude and frequency of spontaneous miniature endplate potentials. Perineural focal recordings of presynaptic currents showed that SNX-230 did not block the neuronal action potential. These and other findings indicated that SNX-230 prevents transmitter release at the mouse neuromuscular junction by blocking calcium channels at presynaptic nerve endings. These calcium channels correspond pharmacologically to VSCCs associated with high-affinity binding sites in rat brain and are most probably either of the P- or Q-type.

Exocytosis at the nerve terminal will only be fully understood when purified synaptic vesicles can be made to fuse with presynaptic plasma membrane in vitro. While the purification of synaptic vesicles from electric organ is now straightforward, isolation of the presynaptic plasma membrane presents a greater difficulty because of lack of specific markers. We have utilized pure synaptic vesicles in a novel way to overcome this difficulty. Antibodies raised to pure synaptic vesicles can be used to detect the presence of vesicle antigens. Thus, for example, we can show that synaptosome preparations isolated from electric organ by conventional procedures have about 5% of their protein in synaptic vesicles. The synaptic vesicle antigens and choline acetyltransferase both disappear from the synaptosome fraction after denervation of the electric organ, verifying that they are nerve terminal specific. Some of the synaptic vesicle antigens can be detected on the outside of the intact synaptosomes by binding rabbit anti-synaptic vesicle antibodies. Such antibody-coated synaptosomes will absorb specifically to goat anti-rabbit IgG attached to polyacrylamide beads. Lysis of such bead-bound synaptosomes leaves a plasma membrane fraction firmly attached to the beads. By raising antibodies to this membrane fraction an antiserum has been generated that binds to antigenic determinants in the electric organ. Some of these antibodies bind to the frog neuromuscular junction. The antigens recognized by this antiserum are, like synaptic vesicle antigens, lost on denervation. Unlike synaptic vesicle antigens, however, the antigens recognized by the anti-plasma membrane serum are present on the outside of resting frog nerve terminals.